Linked Life Data

9632696

Source:http://linkedlifedata.com/resource/pubmed/id/9632696

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
26
pubmed:dateCreated
1998-8-3
pubmed:abstractText
Steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating delivery of cholesterol to cytochrome P450scc on the inner mitochondrial membrane. StAR is synthesized in the cytoplasm and is subsequently imported by mitochondria and processed to a mature form by cleavage of the NH2-terminal mitochondrial targeting sequence. To explore the mechanism of StAR action, we produced 6-histidine-tagged N-62 StAR (His-tag StAR) constructs lacking the NH2-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. In vitro import assays demonstrated that wild-type StAR preprotein was imported and processed to mature protein that was protected from subsequent trypsin treatment. In contrast, His-tag StAR was not imported and protein associated with mitochondria was sensitive to trypsin. Using metabolically labeled COS-1 cells transfected with wild-type or His-tag StAR constructs, we confirmed that wild-type StAR preprotein was imported and processed by mitochondria, whereas His-tag StAR remained largely cytosolic and unprocessed. To determine whether cytosolic factors are required for StAR action, we developed an assay system using washed mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in Escherichia coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import, and in the absence of cytosol. The ability to produce bioactive recombinant StAR protein paves the way for refined structural studies of StAR and StAR mutants.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
26
pubmed:volume
273
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
16339-45
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
The mechanism of action of steroidogenic acute regulatory protein (StAR). StAR acts on the outside of mitochondria to stimulate steroidogenesis.
pubmed:affiliation
Center for Research on Reproduction and Women's Health and the Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't